The invention relates to the synthesis of L-2-amino-4-(hydroxymethylphosphinyl)butyric acid (L-phosphinothricin) from 4-(hydroxymethylphosphinyl)-2-oxobutyric acid (HMPB) by coupling of two enzymatic reactions.
The preparation of phosphinothricin in a coupled enzymatic synthesis has already been disclosed in EP 249 188. This describes the conversion of xcex1-ketoglutarate into glutamate in the presence of the amino donor aspartate and of a glutamate/oxalacetate transaminase (GOT). This conversion is coupled to another transaminase reaction which converts 4-(hydroxymethylphosphinyl)-2-oxobutyric acid (HMPB) into L-phosphinothricin in the presence of the amino donor glutamate formed in the first reaction.
Now, for complete substrate conversion in coupled enzyme reactions, it is important that the equilibrium constants of the individual enzyme reactions differ from one another. However, the equilibrium constant of transamination reactions is about 1.0 so that in general only a 50% yield of the required product is possible (U.S. Pat. No. 4,826,766).
It is possible to displace the equilibrium of enzymatic reactions in favor of the products either by removing one reaction product from the equilibrium or by employing an excess of one starting substance. In general the amino donor glutamate is employed in high excess for the said transamination reaction for synthesizing L-phosphinothricin [A. Schulz et al. (1990) Appl. Environ Micobiol. 56, 1-6, No. 1]. However, this has the disadvantage that the non-proteinogenous amino acid L-phosphinothricin can be separated from the natural amino acids, such as, for example, glutamate, only with great elaboration, for example by two consecutive ion exchange chromatographies [A. Schulz et al. (1990) Appl. Environ. Micobiol. 56, 1-6 No. 1].
An alternative possibility in a coupled process involving a phosphinothricin-specific transaminase and a GOT is for the reaction to be driven by the GOT in the direction of phosphinothricin synthesis by recycling the glutamate consumed by the phosphinothricin transaminase, because the GOT reaction product, oxalacetate, is spontaneously decarboxylated to pyruvate in the presence of doubly charged metal ions and is thus removed from the reaction equilibrium. However, all GOTs hitherto described have a subsidiary activity which transaminates pyruvate to alanine in the presence of glutamate, so that NH4+ is continuously removed from the reaction, and the reaction to give L-phosphinothricin cannot go to completion when equimolar amounts of HMPB and amino donor (glutamate and/or aspartate) are used. In addition, the resulting L-phosphinothricin is contaminated with alanine.
It has now been found that phosphinothricin is produced, in virtually quantitative yield and virtually without any contamination whatever by a natural amino acid, in a coupled enzyme reaction with a transaminase with GOT activity and with a transaminase with L-phosphinothricin transaminase activity when the amino donor glutamate is employed in catalytic amounts and the amino donor aspartate is employed in approximately equimolar amounts relative to HMPB.
Hence the invention relates to:
A process for the preparation of L-2-amino-4-(hydroxy-methylphosphinyl)butyric acid (L-phosphinothricin) of the formula (I) 
from 4-(hydroxymethylphosphinyl)-2-oxobutyric acid (HMPB) of the formula (II) 
in a coupled enzyme reaction comprising the following steps:
a) reaction of aspartate and xcex1-ketoglutarate in the presence of a suitable transaminase 1 to give oxalacetate and glutamate and
b) reaction of glutamate and HMPB of the formula (II) in the presence of a suitable transaminase 2 to give xcex1-ketoglutamate and L-phosphinothricin,
wherein the molar ratio of aspartate to HMPB is 0.5-1.5 to 1, preferably 0.8-1.2 to 1, in particular equimolar, and glutamate or xcex1-ketoglutarate is added in catalytic amounts.
Glutamate, aspartate and xcex1-ketoglutarate or their corresponding acids are substances which can be be bought. HMPB can be prepared as described in EP 30424.
The transaminase 1 is any enzyme which has transaminase activity and is able to transaminate xcex1-ketoglutarate to glutamate in the presence of aspartate as amino donor (so-called enzyme with GOT activity). It is preferable to use a GOT (glutamate/oxalacetate transaminase) which is unable to transaminate pyruvate to alanine and, in this way, would not contaminate the required product of the coupled reaction, L-phosphinothricin, with another natural amino acid. Pyruvate is produced, for example, by decarboxylation of oxalacetate, the transamination product of aspartate.
It is possible to use, in particular, transaminases which have GOT activity, from Escherichia coli (E. coli) or from bacteria of the genus Bacillus, and which have no pyruvate-specific activity.
The transaminase 2 is any enzyme which has transaminase activity and is able to transaminate HMPB to L-phosphinothricin in the presence of glutamate as amino donor. Enzymes of this type are indicated in EP 249 188. It is preferable to use as transaminase 2, in analogy to transaminase 1, a transaminase which is unable to transaminate pyruvate to alanine.
In particular, the L-phosphinothricin-specific transaminase from E. coli has no pyruvate-specific activity and can therefore be used by preference. The L-phosphinothricin-specific transaminase from E. coli can be concentrated, for example, by the method of A. Schulz (1990).
It is possible in principle also to use for the transamination reactions whole cells with the particular transaminase activity, cell extracts, partially purified cell extracts or purified enzymes. However, it is advantageous to employ enzymes which have been purified until subsidiary reactions no longer occur, especially the transamination of pyruvate to alanine.
It is particularly advantageous to employ at least one enzyme, in particular both, in immobilized form. A possible process for the immobilization of transaminases is described, for example, by A. Schulz et al. (1990).
The transamination reactions are generally carried out in a biocompatible buffer, i.e. in a buffer which maintains the pH in a range from 6.5 to 10, preferably 7.5 to 9.0, in particular 7.5 to 8.5, and does not react with the individual reactants. It is preferable to select a phosphate or tris buffer, in particular a tris buffer. The molar ratio of aspartate to HMPB is 0.5-1.5 to 1, preferably 0.8-1.2 to 1, in particular equimolar. Glutamate or xcex1-ketoglutarate is added to the reaction mixture in catalytic amounts. The ratio of glutamate or xcex1-ketoglutarate to HMPB is generally 0.01-1 to 1, preferably 0.01-0.2 to 1, in particular 0.05-0.2 to 1. The reaction mixtures generally also contain small amounts of the cofactor pyridoxal phosphate, for example in a concentration 1-500 xcexcm, preferably 5-100 xcexcm. The reaction temperature is generally between about 20 and 70xc2x0 C., preferably from 30 to 400xc2x0 C.
To increase the yield of L-phosphinothricin it is preferable to decarboxylate in the presence of multiply charged metal ions the oxalacetate produced in the glutamate/oxalacetate transaminase reaction (UK Patent Application 2 161 159). Examples of suitable multiply charged metal ions are all the metal ions listed in the British Patent Application, preferably Al3+, Mg2+, Mn2+, Fe2+ or Fe3+. It is furthermore advantageous in this preferred embodiment to use no transaminase or transaminase fraction with an additional pyruvate-specific activity. The increase in the yield of L-phosphinothricin by the decarboxylation reaction of oxalacetate under the stated reaction conditions was completely unexpected because, according to UK Patent Application 2 161 159, the highest product yield was found only with aspartate as sole amino donor in the reaction system.
The virtually complete consumption of the amino donors aspartate and glutamate results in the product L-phosphinothricin being virtually free of natural amino acids, and separation of it from the formed xcex1-keto acids xcex1-ketoglutarate and oxalacetate or pyruvate is possible in a single step. Although xcex1-ketoglutarate or glutamate has been added not in excess but only in very small amounts and, likewise, aspartate has been added not in excess but preferably equimolar relative to HMPB, nevertheless virtually complete conversion of HMPB into L-phosphinothricin has been observed. This was in no way predictable.
L-phosphinothricin can be purified straightforwardly by known processes, for example by extraction with methyl isobutyl ketone or by cation exchange chromatography, such as, for example, with Amberlite IR 120.
The L-phosphinothricin which is obtained is generally employed in agriculture as herbicide. The following examples are intended to explain the invention in more detail without restricting it in any way.